Pharmacological characterization and antidiabetic activity of a long-acting glucagon-like peptide-1 analogue conjugated to an antithrombin III-binding pentasaccharide.

AIMS: To examine the biological characteristics of a novel glucagon-like peptide-1 (GLP-1) conjugate, in which an antithrombin III (ATIII)-binding pentasaccharide is conjugated to d-Ala(8) GLP-1 using a tetraethylene glycol linker. METHODS: We assessed GLP-1 receptor binding, cAMP generation and insulin secretory activity of the GLP-1 conjugate in vitro. Circulating half-life, glucose homeostatic and subchronic therapeutic effectiveness were then examined in vivo. RESULTS: The half-life of the GLP-1 conjugate in mice was ∼11 h. In vitro insulin secretion from clonal ß cells and islets was increased (p < 0.001) by the conjugate. The conjugate had half maximum effective concentration values of 1.3 × 10(-7) and 9.9 × 10(-8) M for displacement of (125) I-GLP-1 in competitive GLP-1 receptor binding and cAMP generation, respectively. Glucose tolerance in normal mice, immediately and 4 h after conjugate injection, resulted in significant (p < 0.001) improvements in blood glucose. These effects persisted for >48 h after administration. Daily treatment (21 days) of high-fat-fed and ob/ob mice with 25 nmol/kg conjugate resulted in significant improvement in glucose tolerance (p < 0.001) and reductions in glycated haemoglobin (HbA1c; p < 0.01) equivalent to or better than with exenatide or liraglutide. Treatment of C57BL/KsJ db/db mice for 15 days with 100 nmol/kg conjugate significantly (p < 0.001) reduced glucose and raised plasma insulin. Oral glucose tolerance was significantly (p < 0.001) improved and both 24-h glucose profile (p < 0.001) and HbA1c levels (p < 0.001) were reduced. Islet size (p < 0.001) and pancreatic insulin content were increased without change of islet cell proliferation or apoptosis. CONCLUSION: These data show that d-Ala(8) GLP-1(Lys(37) ) pentasaccharide exerts significant antidiabetic actions and has a projected pharmacokinetic/pharmacodynamic profile that merits further evaluation in humans for a possible once-weekly dosing regimen.

Conjugation, immunoreactivity, and immunogenicity of calix[4]arenes; model study to potential calix[4]arene-based Ac3+ chelators.

For the development of calix[4]arene-based radiotherapeutic agents, the conjugation to biomolecules and immunogenicity in mice of potential 225Ac3+-chelating calix[4]arenes were studied. A calix[4]arene triethyl ester isothiocyanate and a bis(calix[4]arene) hexacarboxylic acid, containing a masked thiol functionality, were used in conjugation experiments to a mouse monoclonal antibody and serum albumins. All characterization techniques indicate that only the calix[4]arene carboxylic acid is successfully conjugated to the biomolecules. The immunoreactivity of the conjugates is not impaired when up to 6 equiv of calixarene are bound to the monoclonal antibody. Animal tests indicated that the immunogenicity toward the calix[4]arene is strongly influenced by the nature of the carrier, the dosage, and the injection method. No immune response occurred when a homologous carrier was used or when a heterologous carrier was applied at a dosage of 10 microg per immunization via intravenous injection. Under all other conditions, the presence of antibodies directed against the calix[4]arene was demonstrated. Thus, for the application in radioimmunotherapy, the conjugation of a calix[4]arene to a humanized antibody will probably not lead to an immune response, and the immunoreactivity will not be disturbed.

Electrophoretic method for the quantitative determination of a benzyl-DTPA ligand in DTPA monoclonal antibody conjugates.

A simple electrophoretic IEF procedure was developed for the quantitation of bifunctional DTPA ligand molecules in DTPA-protein conjugates. From a calibration plot of pI versus substitution ratios of reference conjugates, the concentrations of DTPA conjugated to protein were determined. Molar ratios of DTPA to protein agreed satisfactorily with the ratios obtained by a spectrophotometric technique using a colored yttrium(III) complex of arsenazo III. The IEF method was successfully applied on preparations of benzyl-DTPA to mAbs MOPC-21, SC-20 (aCEA), and human serum albumin.

In vitro evaluation of DNA-DNA hybridization as a two-step approach in radioimmunotherapy of cancer.

The specific delivery of radioisotopes to a tumor at minimal radiation of normal tissue is the ultimate aim of radioimmunotherapy. In this respect a two-step pretargeting regimen generally leads to an improved tumor to normal tissue uptake ratio compared to direct administration of radioimmunoconjugates. In this paper, in vitro studies are described in which the specific hybridization of complementary DNA fragments is the recognition mechanism in a pretargeting regimen comprising tumor cell saturation with a monoclonal antibody (MoAb)-oligonucleotide conjugate, followed by administration of the radiolabeled complementary oligonucleotide. Complementary oligodeoxynucleotides (15-mers; melting temperature, 68 degrees C) were prepared on a DNA synthesizer. The 5'-end was derivatized with a functional group for labeling with iodine, and the 3'-end was substituted with an amino function suitable for conjugation to an antibody (or attachment of a biotin residue). Both terminal modifications ensure stability of the oligonucleotides against exonucleases because the unconjugated form is stable for 24 h and the conjugated form is stable for several days when incubated in human plasma at 37 degrees C. Antibody-DNA conjugates were prepared by introduction of sulfhydryl groups into the oligonucleotide, followed by conjugation to maleimide-substituted MoAbs. Typically, 3 oligonucleotides were conjugated to an IgG, and 4-6 were conjugated to an IgM with preservation of immunoreactivity. Histochemistry on fresh frozen sections of breast cancer tissue demonstrated qualitatively the specificity of our two-step procedure. In vitro experiments with human tumor cell lines and tumor-specific MoAbs showed that, after saturation with tumor-specific MoAb-DNA conjugates, quantitative hybridization of the tumor cell-bound oligonucleotides occurred at a 30-fold excess of the labeled complementary oligonucleotide: hybridization was complete after 30 min of incubation. No reaction was observed with an irrelevant MoAb-DNA conjugate. The oligonucleotide was neither taken up by tumor cells or endothelial cells nor hybridized to a significant extent with human genomic DNA. These data indicate the feasibility of this two-step approach in radioimmunotherapy.

Specific recognition of antibody-oligonucleotide conjugates by radiolabeled antisense nucleotides: a novel approach for two-step radioimmunotherapy of cancer.

One of the major challenges in radioimmunotherapy is the specific delivery of radioisotopes to tumor cells while minimizing normal tissue radiation. In this respect, the application of two-step pretargeting schemes generally leads to more favorable tumor to normal tissue uptake ratios than direct administration of radioimmunoconjugates. In this study, we present the specific hybridization of complementary DNA fragments as a novel recognition mechanism in pretargeting. Briefly, our strategy involves first administration of antibody-DNA conjugate, followed by targeting with radiolabeled complementary DNA (antisense DNA). Complementary oligodeoxynucleotides (14-mers, Tm = 57 degrees C), in which part of the phosphodiesters has been replaced by methylphosphonates (to ensure stability against nucleases), were prepared on a DNA synthesizer. The oligonucleotides were further derivatized via a uridine moiety at their 5'-end in such a way that radiolabeling or conjugation with antibodies could be accomplished. Both a murine IgG (anti-hCG) and the human anti-tumor IgM 16.88 were conjugated with one to three oligonucleotides via the heterobifunctional cross-linker SMCC. Incubation of these immunoconjugates with the radiolabeled antisense DNA revealed specific hybridization with the antibody-linked oligonucleotides. Antigen binding studies performed with antigen-coated matrices showed that the immunoreactivity of the antibody-DNA conjugate is preserved. Moreover, it is demonstrated that the radiolabeled DNA is still capable of hybridizing selectively with the oligonucleotides of the immunoconjugate, when the latter is bound to its antigen.

A novel assay for the quantitative determination of lipoprotein-X (LP-X) in serum.

A new, fast and simple quantitative LP-X assay is described. The method selectively removes apoB-containing lipoproteins by precipitation with specific antibodies. To the supernatant, a precipitation system for LP-X, viz. SDS and MgCl2 in final concentrations of 0.5 g/l and 0.2 mol/l respectively, is added and the turbidity measured at 360 nm. The assay is linear from 0.2 g/l-5.0 g/l and shows a high precision (inter-assay CV of less than 3%). The test correlates favourably with other quantitative LP-X assays, but has the particular advantage that it can be automated and that the time required for one series is only 2 h.

3,3',5,5' - Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay.

The use of 3,3',5,5' - tetramethylbenzidine as non-mutagenic chromogen for the end point determination in enzyme-immunoassay (EIA) is described. In sandwich EIAs for HCG and HBsAg and in a competitive EIA for testosterone, the colour yield with TMB was superior to that obtained with o-phenylene diamine (OPD), which was by far the best chromogen for horse-radish peroxidase until now. This led to an improvement of sensitivity and precision of the assays and makes EIA even more competitive with other types of immunoassays.

Hormonal control of vitellogenin synthesis in avian liver.

Estradiol induces the synthesis of vitellogenin in the avian liver. We describe the precursor-product relationship between vitellogenin and the yolk proteins phosvitin and lipovitellin. The high rate of vitellogenin synthesis is a consequence of the accumulation of a stable messenger RNA. We suggest that estradiol acts at the level of the genome by opening a hitherto non-transcribed gene.